Human metapneumovirus (HMPV) was first reported in 2001 and has quickly come to be recognized as a significant agent of respiratory tract disease worldwide, especially in the pediatric population, in immunocompromised individuals, and in the frail elderly. We are using recombinant DNA methods to characterize viral molecular biology and pathogenesis and to develop attenuated derivatives of HMPV for use as a live intranasal pediatric vaccine.[unreadable] HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA of approximately 13.3 kb. We previously developed the first complete HMPV consensus sequences representng the two genetic subgroups, A and B. Molecular studies by ourselves and others showed that HMPV encodes eight mRNAs that are translated into nine proteins (the M2 mRNA contains two separate overlapping open reading frames encoding two distinct proteins, M2-1 and M2-2). By analogy to human respiratory syncytial virus, its better-known relative, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; M2-1, RNA synthesis factor; M2-2, RNA synthesis factor; SH, small hydrophobic protein; G, attachment glycoprotein; and L, viral polymerase.[unreadable] We developed a reverse genetic system for HMPV whereby complete infectious virus can be generated in cell culture entirely from cloned cDNAs. This provides a method for engineering the genome in pursuit of basic studies and for designing vaccines. We found that four viral genes could be deleted individually and in various combinations with little or no effect on viral replication in vitro, namely: G, SH, M2-1 and M2-2. Evaluation of the attenuation and immunogenicity of these viruses in hamsters and African green monkeys indicated that the del-G virus (with or without the additional deletion of the SH gene) and del-M2-2 virus are promising candidates to be live attenuated vaccines against HMPV. [unreadable] Additional vaccine candidates were generated by replacing the N or P open reading frame of HMPV with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. AMPV is attenuated in primates due to a natural host range restriction, and it was hoped that the chimeric viruses would have a host range restriction due to the AMPV N or P gene. Evaluation in hamsters and African green monkeys showed that this indeed was the case, and the HMPV-AMPV-P virus in particular is a promising vaccine candidate.[unreadable] Preparations of the del-M2-2, and HMPVAMPV-P viruses have been produced under conditions appropriate for products for human use and will be amplified to make clinical trial material for phase I studies of safety, attenuation and immunogenicity in human volunteers. The P chimera is an attractive candidate because it combines improved growth in vitro that is characteristic of AMPV with attenuation in vivo. The del-M2 virus is attractive because M2-2 appears to be involved in regulating viral RNA synthesis, and its deletion up-regulates gene expression resulting in increased antigen synthesis. [unreadable] Analysis of intracellular viral mRNAs made in response to virus lacking the M2-1 protein showed that they contained polyA tails that were approximately 25 nucleotides in length compared to the 150-250-nucleotide length that is characteristic of eukaryotic mRNAs. This was confirmed with a mini-replicon system. Thus, HMPV M2-1 appears to have a role in the synthesis or stability of the polyA tail. This is being further investigated.[unreadable] Northern blot analysis of intracellular HMPV RNA from infected cells showed that the various viral genes differ considerably with regard their representation in monocistronc mRNA versus readthough mRNAs. Studies with a mini-replicon system showed that differences in the efficiency of termination appeared to be due completely to differences in the gene-end (GE) signal (and in particular does not involve the various intergenic and gene-start signals). The GE signals of the M2 and SH genes are particularly inefficient, resulting in very low levels of expression of the respective downstream SH and G genes. Alignment of the sequences of the various GE signals indicated that they usually differ by only one or two nucleotides, indicating that these differences presumably are the basis for the functional differences. [unreadable] We evaluated the biological significance of these inefficient signals by making recombinant viruses in which an efficient signal (in the M gene) and an inefficient signal (in SH) were swapped in various combinations. Any change from the natural arrangement was associated with a decrease in plaque size in vitro, although there was essentially no effect on virus yield. Any change from the natural arrangement also was associated with decreased replication in hamsters. The greatest effect on replication in hamsters was observed with any arrangement in which the GE signal of the M gene was inefficient. This effect presumably was due to reduced expression of the next-downstream F gene. These studies provided the first demonstration that differences in the efficiency of the GE signals appear to confer an optimal ratio of expression of the various viral genes, such that changes result in suboptimal replication in vivo. [unreadable] During the preparation of a number of recombinant HMPVs, consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This was suggestive of sizable subpopulations containing mutations. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There also were two instances of nucleotide deletion and numerous instances of nucleotide substitution point mutations, mostly in the SH gene. Analysis of molecularly cloned cDNAs derived from these preparations confirmed that the additional peaks on the sequencing electropherograms indeed represented subpopulations of molecules containing the various changes. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequencing.[unreadable] The occurrence of mutant subpopulations was greatly reduced by substitution of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. The fact that SH is dispensable for replication in vitro likely in one factor in this high frequency of recovered mutations. However, this is not sufficient explanation, since G also is dispensable but accumulated far fewer mutations. In addition, most of the observed mutations occurred in the SH open reading frame and were such that they ablated expression of SH by frame-shifts. Thus, there appeared to be a preferential accumulation of mutations silencing SH, suggesting that this conferred a (modest) growth advantage in vitro (but not in vivo, since silencing of SH does not seem to occur in nature). In any event, these results indicate the need to carefully monitor vaccine virus by sequence analysis and to proactively remove mutational hot spots, which usually involve A or U tracts in sequence that is non-essential for replication in vitro.